Project Summary Project 3 Despite overexpression of the epidermal growth factor receptor (EGFR) on nearly all head and neck squamous cell carcinomas (HNSCC), the EGFR-specific monoclonal antibody (mAb) cetuximab is effective only in a minority of patients. The modest effects of cetuximab have stimulated interest in determining its anti-tumor mechanisms and the factors that limit clinical responses, in order to improve its efficacy by combining immunotherapeutic approaches. A growing body of evidence, and our preliminary results, indicate that a tumor antigen (TA)-specific mAb, such as cetuximab, can effectively trigger TA-specific cytotoxic T lymphocyte (CTL) responses. However, these effector CTLs are not fully effective, raising the possibility that inhibitory mechanisms limit the efficacy of cetuximab in the majority of patients. Blockade of these inhibitory mechanisms could restore anti-tumor activity and enhance cetuximab efficacy. However, the inhibitory mechanism(s) that are primarily responsible for limiting cetuximab efficacy remain unknown. A high frequency of peripheral blood T-lymphocytes (PBL) and tumor infiltrating T-lymphocytes (TIL) in multiple solid tumors, including HNSCC, express elevated levels of inhibitory receptors (so-called ?checkpoints?), such as cytotoxic T lymphocyte antigen (CTLA-4) and programmed death-1 (PD-1), rendering them unresponsive to antigenic stimulation (exhaustion). Furthermore, CTLA-4+ regulatory T cells (Tregs), a potently suppressive sup- population of CD4+ T cells that produce TGF-?, are often present at increased frequencies and with enhanced suppressive capacity in tumors. Collectively, inhibitory receptors on CTL and Tregs are major barriers to effective anti-tumor T cell responses, which has been demonstrated by striking clinical responses to anti- CTLA-4 and anti-PD-1 antibodies. Our previous studies and preliminary data support a working model in which the generation of TA-specific CD8+ T cells triggered by cetuximab in the non-responder patient population occurs concurrently with increased inhibitory mechanisms (including TGF-?) that reduce the clinical response to cetuximab therapy. This project will take advantage of the availability of biospecimens from two novel UPCI clinical trials, which have either recently completed (UPCI 08-013, testing immune biomarkers in single-agent cetuximab treated patients)) or are ongoing (UPCI 12-084, combining cetuximab with anti-CTLA-4 mAb ipilimumab to inhibit Treg). We will determine whether inhibitory mechanisms are responsible for limiting anti-tumor activity induced in cetuximab-treated HNSCC patients and directly assess the impact of CTLA4+ Tregs in this new, ongoing clinical trial.